to converging on a common subset of opine
synthesis genes (Fig. 4B).
The structural arrangement of modules
affects the degree of flexibility of oncogenic
plasmid types. Most T-DNAs have a gene for
synthesizing an opine; opines are conjugates
between a keto acid and an amino acid (K-A),
a sugar and an amino acid (S-A), or two sugars
(S-S). All oncogenic plasmids have at least one
T-DNA with an opine synthesis gene, and plas-
mid classification traditionally relied on the
opine synthesized, despite assumptions of fre-
quent exchange of opine genes (Fig. 4). Al-
though variation of opine genes does exist,
there is no evidence for the generalization of
rampant exchange among all types of plas-
mids (Fig. 4C). Opine variation is influenced
by the proximity of genes to the right borders
of T-DNAs. When the cognate opine anabo-
lism and catabolism/uptake genes are closely
linked and separated by only the right border,
swapping of opine loci and the border se-
quence can occur with little constraint (figs.
S5, S6, S14, S17, and S18). Type I.b Ti plasmids
are very promiscuous (fig. S5). We hypothe-
size that swapping is mediated by a process
that involves nonhomologous recombination
within oncogenes. In type III Ti plasmids,
exchange is predicted to be mediated by ho-
mologous recombination among oncogenes
(fig. S6). Because K-A and S-A genes are as-
sociated with different sets of oncogenes,
swapping is most frequently limited to with-
in their structural groups of opines (fig. S17and data S6). Type I.a Ti plasmids are struc-
turally similar to type I.b Ti plasmids and
have the potential to diversify opines but are
far more limited. We suggest that the low
diversity is a consequence of the ancestral
type I.a Ti plasmid experiencing a bottleneck
along with BV2 (Fig. 1).
In other types of plasmids, important func-
tional modules separate opine genes, and
multiple recombination events would need
to occur to acquire the two interdependent
modules. For example, opine genes of type II
plasmids are interrupted bytragenes (fig. S6).
In type IV.a Ti plasmids, genes necessary for
anabolism and catabolism of nopaline are
separated by those involved in the catabo-
lism of a second opine, agrocinopine (fig. S7).
Theacsgene, cognate toaccand necessary
for synthesizing agrocinopine, is within and
adjacent to the left border of the T-DNA (Fig.
4B and fig. S13). This organization is restrictive;
for opine swapping to occur, either a larger
fragment that included most of the T-DNA
would also need to be exchanged, or the two
modules for nopaline would need to be ac-
quired separately.
Thetrblocus is associated with a high num-
ber of single-nucleotide polymorphisms (SNPs)
and has diverse alleles (figs. S3 to S9 and S19).
These data are consistent with the possibil-
ity of recombination within thetrblocus, and
becausetrbis adjacent to opine loci, it likely
contributes to the swapping of opine cassettes
in many types of Ti plasmids. Some type III Ti
plasmids have additional copies oftrbgenes
located distal to the maintrblocus, likely due
to duplication resulting from recombination
within the locus (fig. S18B). In contrast, in
the Ri class,trbis paired withtraand is sep-
arated from opine loci by large regions with
no genes involved in virulence or plasmid
maintenance (fig. S9). This difference in spatial
relationship between classes likely constrains
recombination between Ti and Ri plasmids.
However,traandtrbcould mediate recombi-
nation withhomologous loci of the diverse
non-oncogenic plasmids that are prevalent in
the ARC (fig. S20).
All findings were synthesized to model the
evolution of oncogenic plasmids (Fig. 5). Ti
and Ri plasmids are distantly related and are
hypothesized to have emerged from an ances-
tral proto-oncogenic plasmid ( 13 , 26 ). We hy-
pothesize that this ancient replicon carried
vir,tzs,acs, and an opine synthesis gene
flanked by T-DNA borders (fig. S21 and data
S8). Thetzsgene is a paralog ofipt(tmr) that
is distal to the T-DNA and not transferred
into plant cells ( 27 ). Thetzsandiptgenes en-
code isopentenyltransferases, enzymes that
catalyze the first step in the synthesis of the
plant growth–promoting hormone cytokinin
( 28 ). However, cytokinins derived from Tzs
have been implicated in regulatingvirgeneWeisberget al.,Science 368 , eaba5256 (2020) 5 June 2020 4of8
Fig. 3. Variations within and between types and subtypes of oncogenic plasmids.(A) Visualization of
variation within plasmid subtypes. Circles, starting from the innermost circle, are a gene synteny graph, a
plasmid map, and bar graphs representing relative depth of coverage of sequencing reads (sliding window of
1 kb), number of SNPs (sliding window of 1 kb), and number of soft-clipped reads (≥5 clipped reads). Nodes
in the synteny graph represent genes present in≥1 plasmid of a type. Nodes are connected if genes are
adjacent in at least one plasmid. The network cycle represents the most common order of genes within a
type. Different structures, such as gene presence/absence variation, rearrangements, or inversions present in
plasmids, are shown as alternative paths in the cycle. Nodes are colored according to key functions and
aligned to corresponding features in the plasmid map, or colored yellow for transposase- and insertion
sequence–encoding genes. Soft-clipped reads are those that must be trimmed in order to align to the
reference and can be evidence for a recombination breakpoint. Type I.a and type I.b Ti plasmids are
presented as examples. (B) General structure and organization of type II–VI Ti plasmids, as well as their
subtypes, and type I–III Ri plasmids. Simplified maps show locations and spatial relationships of key modules.
They are colored and labeled with a letter code: A,acc; O, opine genes; R,repABC; T-1 to T-4, T-DNA 1–4;
Ta,tra;Tb,trb;V,virgenes. The triangle represents an insertion sequence. Visualizations are as shown
in (A) for each of the plasmid types and subtypes (figs. S5 to S9).
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